Affiliations

1 FAS Center for Systems Biology, Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachusetts, United States of America; Broad Institute, Cambridge, Massachusetts, United States of America.

8 Department of Pathology, University of Texas Medical Branch, Galveston, Texas, United States of America.

9 FAS Center for Systems Biology, Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachusetts, United States of America; Broad Institute, Cambridge, Massachusetts, United States of America; Department of Immunology and Infectious Disease, Harvard School of Public Health, Boston, Massachusetts, United States of America.

Affiliations

1 FAS Center for Systems Biology, Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachusetts, United States of America; Broad Institute, Cambridge, Massachusetts, United States of America.

8 Department of Pathology, University of Texas Medical Branch, Galveston, Texas, United States of America.

9 FAS Center for Systems Biology, Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachusetts, United States of America; Broad Institute, Cambridge, Massachusetts, United States of America; Department of Immunology and Infectious Disease, Harvard School of Public Health, Boston, Massachusetts, United States of America.

Abstract

Next-generation sequencing (NGS) has the potential to transform the discovery of viruses causing unexplained acute febrile illness (UAFI) because it does not depend on culturing the pathogen or a priori knowledge of the pathogen's nucleic acid sequence. More generally, it has the potential to elucidate the complete human virome, including viruses that cause no overt symptoms of disease, but may have unrecognized immunological or developmental consequences. We have used NGS to identify RNA viruses in the blood of 195 patients with UAFI and compared them with those found in 328 apparently healthy (i.e., no overt signs of illness) control individuals, all from communities in southeastern Nigeria. Among UAFI patients, we identified the presence of nucleic acids from several well-characterized pathogenic viruses, such as HIV-1, hepatitis, and Lassa virus. In our cohort of healthy individuals, however, we detected the nucleic acids of two novel rhabdoviruses. These viruses, which we call Ekpoma virus-1 (EKV-1) and Ekpoma virus-2 (EKV-2), are highly divergent, with little identity to each other or other known viruses. The most closely related rhabdoviruses are members of the genus Tibrovirus and Bas-Congo virus (BASV), which was recently identified in an individual with symptoms resembling hemorrhagic fever. Furthermore, by conducting a serosurvey of our study cohort, we find evidence for remarkably high exposure rates to the identified rhabdoviruses. The recent discoveries of novel rhabdoviruses by multiple research groups suggest that human infection with rhabdoviruses might be common. While the prevalence and clinical significance of these viruses are currently unknown, these viruses could have previously unrecognized impacts on human health; further research to understand the immunological and developmental impact of these viruses should be explored. More generally, the identification of similar novel viruses in individuals with and without overt symptoms of disease highlights the need for a broader understanding of the human virome as efforts for viral detection and discovery advance.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1. Viruses identified in UAFI samples…

Fig 1. Viruses identified in UAFI samples and afebrile controls.

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( A ) Overview of…

Fig 1. Viruses identified in UAFI samples and afebrile controls.

(A) Overview of reads we identified in Illumina HiSeq reads from 94 singleton UAFI samples and 34 apparently healthy singleton control samples. For all samples, we removed human reads and common bacterial contaminants, and subjected the remaining reads to BLASTn and BLASTx queries of GenBank and assigned to taxonomic kingdoms using MEGAN 4. (B) Viruses identified in singleton (RNA-seq libraries constructed from a single individual) and pooled libraries (RNA-seq libraries constructed from several individuals). In the case of pooled libraries, the percentage refers to the number of libraries, not individual samples.

(A) Overview of the data generated for each novel rhabdovirus. (B) A schematic showing the assembled genomes, consisting of the following genes: nucleoprotein (N), phosphoprotein (P), matrix (M), U1/U2/U3 (uncharacterized accessory proteins), glycoprotein (G), and polymerase (L). We indicate in orange (EKV-1) and blue (EKV-2) segments of the viral genomes that could not be assembled from Illumina reads and instead Sanger sequenced. (C) Coverage plots of the final viral genomes.

Fig 3. Phylogenetic analysis of rhabdovirus polymerase…

Fig 3. Phylogenetic analysis of rhabdovirus polymerase proteins.

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We created a Bayesian phylogenetic tree using…

Fig 3. Phylogenetic analysis of rhabdovirus polymerase proteins.

We created a Bayesian phylogenetic tree using full-length polymerase (L) proteins obtained from GenBank. (A) Tree based on alignments of the tibroviruses and ephemeroviruses. Posterior probabilities are shown at relevant nodes (scale bar = nucleotide substitutions/site) and the tree was midpoint rooted. (B) ELISA detection of EKV-1 and EKV-2 IgG in cohorts from Nigeria and the United States (US normals). Each circle correspond to the raw OD450 value of an individual sample. The mean +/- standard deviation (SD) is shown. Black dotted line = cut-off values for positive samples based on the mean of US normals plus 5x or 3x SD. P-values were calculated using a two-tailed Mann-Whitney test.

Fig 4. Examples of rhabdoviruses reported in…

Fig 4. Examples of rhabdoviruses reported in Africa.

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A map depicting examples of rhabdoviruses isolated…

Fig 4. Examples of rhabdoviruses reported in Africa.

A map depicting examples of rhabdoviruses isolated in sub-Saharan Africa. This map does not depict the current distribution of rhabdoviruses in Sub-Saharan Africa, nor is it meant as a comprehensive listing of all rhabdoviruses isolated in Africa; rather its purpose is to illustrate that many rhabdoviruses have been discovered throughout Africa over the past half-century. Country refers to the sample’s country of origin. Abbreviations: CAR, Central African Republic; DRC, Democratic Republic of Congo.